4.3 DNA Sequencing

Determination of precise sequence of nucleotide in sample DNA

Maxam-Gilbert base destruction method

bases DNA selectively destroyed

not use anymore (reagent toxic&dangerous)

used purified DNA

labelling 5' end (add gamma 32P ATP)

break into G, A+G, C, C+T

Screenshot 2023-12-04 062319

Sanger Method

by Frederick Sanger in 1980

DNA sequence as single strand (by heat)

template DNA supplied w mix of all dNTPs, primer sequence complement to 3' end

diff tube of all four dideoxynucleotide present in limit quantities and label w tag that fluorescence

chain elongation cut off after DNA polymerase insert ddNTP instead of dNTPs

chain termination, primer has defined length of 32P at 5' end and hybridize at 3' end of template strand

generate a series of fragment then use to read the nucleotide sequence

running the product in each tube-on a gel allow to determine each chain terminating ddNTPs was incorporated

stop synthesis selectively at any four ddNTPs

produce a population of molecule

separated by length from the longest to shortest

diff of one nucleotide is enough to separate from other

each ddNTPs light a diff color when illuminated by a laser beam